Undecapeptides containing cysteinesulfinic and cysteinesulfenic acid

ABSTRACT

Disclosed are peptides represented by IVC 1 SLC 2 SC 3 TAW and C 1 SLC 2 SC 3  wherein I stands for isoleucine, V for valine, C 1  for cysteine, C 2  for cysteinesulfinic acid, C 3  for cysteinesulfenic acid, S for serine, L for leucine, T for threonine, A for alanine, and W for tryptophan. These peptides can impart photoreactivity to a protein by binding to a non-heme iron. Also disclosed are methods for imparting photoreactivity to cells by introducing the peptide sequences into at least one protein which is involved in a metabolic system and/or energy metabolic system of the cells. There can be provided peptide sequences with a shorter peptide chain capable of imparting photoreactivity, which can be easily introduced into a protein with a little risk in degrading original function of the protein. There are also provided methods enabling control of metabolic reactions by imparting photoreactivity to cells with the peptides.

This a divisional of application Ser. No. 09/113,312, filed Jul. 10,1998, now U.S. Pat. No. 6,268,475.

FIELD OF THE INVENTION

The present invention relates to a peptide which can impartphotoreactivity to a protein, and a method for controlling metabolism ofcells by introducing a sequence of the peptide into at least one proteinwhich functions in a metabolic system so that the protein should acquirephotoreactivity, thereby controlling metabolism of cells.

BACKGROUND OF THE INVENTION

Nitrile hydratase (NHase) is an enzyme which is produced inmicroorganisms and converts a nitrile compound into an amide compound byhydration. It is a soluble metalloprotein containing iron or cobalt atomin its active center. NHase derived from Rhodococcus sp. N-771 strainhas a non-heme iron center of mononuclear low-spin six coordinateFe(III). NHases have been isolated from several kinds of bacterialcells, and all of them consist of two kinds of subunits, α and β. Bothof the subunits have a molecular weight of about 23,000. NHases ofRhodococcus sp. N-771, N-774, and R312 are considered to be the sameenzyme because their base sequences are identical to each another, andtheir enzymatic activity varies with light irradiation. Namely, whenbacterial cells exhibiting high activity are left in the dark, theenzyme activity is reduced, and the activity is increased again byphoto-irradiation.

It was recently revealed that photodissociation of nitric oxide (NO)which is bound to the non-heme iron center of inactive form Nhaseactivates the enzyme (Odaka et al., J. Am. Chem. Soc., 119, 3785-3791(1997)). However, because the structure of the non-heme iron centerwhich is the photoreactive site was not elucidated yet, the detailedmechanism of the photoreaction remained unclear. Therefore, the presentinventors performed structural analysis of the non-heme iron center, andreported the results (for example, see Protein, Nucleic acid, Enzyme,Vol.42, No.2, p38-45 (1997)). That is, the present inventor isolated theα and β subunits from the inactive form enzyme under denaturationcondition, and found that the NO-binding type non-heme iron center ispresent on the α subunit. Therefore, they further performed enzymaticdegradation of the α subunit with trypsin, and purified the resultingpeptides by reversed phase chromatography under a neutral condition. Asa result, a peptide consisting of 24 residues of ₁₀₅N to ₁₂₈K bindingone iron atom and NO has been isolated. This region was well conservedin various kinds of NHases, and contained a cysteine cluster predictedto be a metal-binding site (₁₀₉C-S-L-₁₁₂C-S-₁₁₄C).

By the way, development of processes for producing useful biomoleculessuch as amino acids, peptides, proteins, carbohydrates, lipids and thelike by utilizing metabolic systems of cells including energy metabolicsystems has been performed for a long time. However, in conventionalmetabolically controlled fermentation, fermentation system for theobjective product is constructed through trials on screening andconcentration of variants resistant to an analogue having a structureanalogous to an objective product. Further, to control the constructedfermentation system, physical factors such as pH and temperature andchemical factors such as substrate concentration and addition ofinducers must be changed. Therefore, various regulatory mechanisms ofthe living body are often affected, and many parameters must bedetermined for optimization of the production scheme. Moreover,regulatory methods of this type have a drawback that it takes a longperiod of time to obtain a reaction to stimulation. Therefore, if it ispossible construct a method for controlling metabolism which enablesproper turning on and turning off a desired intracellular metabolicsystem so as to produce a desired product in a necessary amount when itis required, it will greatly contribute to fermentation processes.

Accordingly, the present inventors studied out a method for artificiallycontrolling metabolism of cells including energy metabolism by utilizingthe peptide stably binding a non-heme iron, which is the photoreactivesite of NHase mentioned above. That is, such a peptide as mentionedabove is introduced into a protein which functions in a metabolic systemof an objective product to impart photoreactivity to the protein so thatthe metabolic system can be controlled by presence or absence of lightirradiation. In this method, photo-control of activity is realized bymodifying a protein which functions in a specific reaction system in ametabolic pathway of an objective product. Because the protein to bemodified can be arbitrarily selected depending on the purpose, themethod has advantages that the screening step by trial and error like inthe conventional method does not required, and that fermentation systemof which metabolism is controlled can be precisely constructed.Furthermore, because activation of enzyme is achieved byphotostimulation, fermentation operation can be performed more simplyand quickly compared with the conventional methods.

However, when such a peptide sequence as mentioned above is introducedinto various kinds of proteins working in intracellular substancemetabolic systems or energy metabolic systems to impart photoreactivityto the metabolism or the energy metabolism, a relatively large peptidelike the aforementioned peptide of 24 residues might have problems. Theproblems are that efficient reaction could not be obtained, orintroduction of the peptide impairs the original function of the labeledprotein in high possibility, because the relatively large proteincontains a large fraction of sequence other than the minimum portionessential for the photoreaction.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to provide a peptidesequence capable of efficient photoreaction, which is a peptide chain ofa minimum unit capable of imparting photoreactivity, not likely toimpair an original function of a labeled protein, and a method forenabling control of metabolic reaction by utilizing the peptide toimpart photoreactivity to cells.

The present invention relates to a peptide having a sequence [SEQ IDNO:3] represented by the following general formula (1).

X₁X₂C₁X₃X₄C₂SC₃X₅X₆X₇  (1)

wherein X₁ to X₇ represents an arbitrary amino acid, C₁ representscysteine, C₂ represents cysteinesulfinic acid, C₃ representscysteinesulfenic acid, and S represents serine.

An embodiment of the present invention is the aforementioned peptidewherein the sequence [SEQ ID NO:4] represented by the general formula(1) is represented as IVC₁SLC₂SC₃TAW wherein I represents isoleucine, Vrepresents valine, C₁ represents cysteine, C₂ representscysteinesulfinic acid, C₃ represents cysteinesulfenic acid, S representsserine, L represents leucine, T represents threonine, A representsalanine, and W represents tryptophan.

Another embodiment of the present invention is the aforementionedpeptide wherein X₁ to X₇ in the sequence represented by the generalformula (1) are selected from amino acids which can maintain higherorder structure of a peptide represented by IVC₁SLC₂SC₃TAW in nitrilehydratase derived from Rhodococcus sp.N-771.

A further embodiment of the present invention is a peptide having asequence represented by the following general formula (2):

C₁X₃X₄C₂SC₃  (2)

wherein X₃ and X₄ represent arbitrary amino acids, C₁ representscysteine, C₂ represents cysteinesulfinic acid, C₃ representscysteinesulfenic acid, and S represents serine. Examples of the abovepeptide include, for example, the aforementioned peptide wherein thesequence represented by the general formula (2) is represented asC₁SLC₂SC₃ wherein C₁ represents cysteine, C₂ represents cysteinesulfinicacid, C₃ represents cysteinesulfenic acid, S represents serine, and Lrepresents leucine. Examples of the above peptide further include, forexample, the aforementioned peptide wherein X₃ and X₄ in the sequencerepresented by the general formula (2) are selected from amino acidswhich can maintain higher order structure of a peptide represented byC₁SLC₂SC₃ in nitrile hydratase derived from Rhodococcus sp. N-771.

The peptides of the present invention can be a peptide which can impartphotoreactivity to a protein by binding a non-heme iron, or a peptidewhich can form a claw setting structure by binding a non-heme iron.

The present invention also relates to a method for impartingphotoreactivity to a cell by introducing one of the aforementionedpeptide sequences of the present invention into at least one proteinwhich is involved in a metabolic system and/or energy metabolic systemof the cell.

In the method of the present invention, together with the peptidesequence, a non-heme iron binding to the peptide can be introduced.

The present invention also relates to a cell having an NO producingsystem and photoreactivity wherein one of the aforementioned peptidesequences of the present invention is introduced into at least oneprotein which is involved in a metabolic system and/or energy metabolicsystem of the cell.

The cell of the present invention may be, for example, a cell introducedwith the peptide sequence and a non-heme iron binding to the peptide, ora cell wherein the peptide sequence and the non-heme iron form a clawsetting structure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents results of reverse phase HPLC of products fromdigestion with thermolysin, carboxypeptidase-Y and leucineaminopeptidase-M.

FIG. 2 is a UV absorption spectrum of a peak at retention time of 11.6minutes in reverse phase HPLC of products from the digestion withthermolysin, carboxypeptidase-Y and leucine aminopeptidase-M.

FIG. 3 shows a claw setting structure formed by a peptide C₁SLC₂SC₃binding to a non-heme iron. In this figure, a binding NO molecule isalso shown.

FIG. 4 shows a hetero-tetramer structure ((αβ)₂) in an asymmetric unit.

FIG. 5 shows omit-annealed Fo-Fc maps of an active site region.

FIG. 6 shows a claw setting structure formed by a peptide C₁SLC₂SC₃binding to a non-heme iron.

FIG. 7 shows an electrospray mass spectrum.

DETAILED DESCRIPTION OF THE INVENTION

As described above, by enzymatically degrading with trypsin the αsubunit of NHase having photoreactivity similar to that of wild typeNHase and binding an NO-binding type non-heme iron center, a peptidecomplex consisting of 24 residues of ₁₀₅N to ₁₂₈K which is binding oneiron atom and NO per one peptide can be isolated. The present inventorsfurther digested the above peptide of 24 residues with thermolysin,leucine aminopeptidase-M and carboxypeptidase-Y, and found a peptidewhich is composed of 11 residues, forms a stable complex with a non-hemeiron, and is a minimum unit for imparting photoreactivity. This peptideis a peptide represented as IVC₁SLC₂SC₃TAW wherein I representsisoleucine, V represents valine, C₁ represents cysteine, C₂ representscysteinesulfinic acid, C₃ represents cysteinesulfenic acid, S representsserine, L represents leucine, T represents threonine, A representsalanine, and W represents tryptophan.

The present inventors further examined the structure of the activecenter of the above peptide by X-ray crystallographic analysis. As aresult, it was found that the 6-residue C₁SLC₂SC₃ containing C₁, C₂, Sand C₃ formed a stable complex directly with the non-heme iron.

Mass spectrometry analysis was performed for the 24-residue peptide inorder to obtain its microstructural information. When mass of thepeptide was determined under light irradiation, the obtained mass waslarger than its theoretical molecular weight by 32 Da. All of the aminoacid residues of this sequence except for the three cysteine residueshad been determined by analysis with a protein sequencer. Therefore, thepeptide was subjected to reduction and then carboxymethylation, andanalyzed by the protein sequencer. As a result, only the 112nd cysteinewas not carboxymethylated. Therefore, the obtained peptide was subjectedto second digestion with thermolysin, and then degraded withaminopeptidase for amino acid analysis. The 112nd cysteine was detectedas the same peak as commercially available cysteinesulfinic acid, andthus it was confirmed that this residue existed as sulfinic acid. Incontrast, any cysteine residue was not modified in an α subunit whichwas expressed as a recombinant in E. coli, and it has been revealed thatthe 112nd cysteine is specifically modified to sulfinic acid inRhodococcus sp. N-771.

In the aforementioned peptide, C₃ represents cysteinesulfenic acid, andit was confirmed that C₃ is cysteinesulfenic acid by analysis of thepeptide digested with trypsin using Fourier transform ion cyclotronresonance mass spectrometry (FT-ICR MS).

The present invention further relates to a peptide having a sequencerepresented by the general formula (1). This peptide was defined basedon the aforementioned sequence IVC₁SLC₂SC₃TAW with a consideration ofthe binding of this peptide and iron atom.

X₁X₂C₁X₃X₄C₂X₅X₆X₇  (1)

In the formula, X₁ to X₇ represent arbitrary amino acids, but inparticular they are suitably selected from amino acids which canmaintain the higher order structure of the above sequenceIVC₁SLC₂SC₃TAW.

The present invention also relates to a peptide having a sequencerepresented by the general formula (2). This peptide was defined basedon the aforementioned sequence C₁SLC₂SC₃ with a consideration of thebinding of this peptide and iron atom.

C₁X₃X₄C₂SC₃  (2)

In the formula, X₃ and X₄ represent arbitrary amino acids, but inparticular they are suitably selected from amino acids which canmaintain the higher order structure of the above sequence C₁SLC₂SC₃.

In particular, the above sequences IVC₁SLC₂SC₃TAW and C₁SLC₂SC₃ arebound to a non-heme iron to form the claw setting structure with itrepresented in FIG. 3. C₁(Cys 109), C₂ (Cys-SO₂H 112), S (Ser 113) andC₃ (Cys-SOH 114) coordinate to the non-heme iron. In the figure, a boundNO molecule is also represented.

Accordingly, X₁ to X₆ in the sequence represented by the general formula(1), and X₃ and X₄, in the sequence represented by the general formula(2) are suitably selected from amino acids which can bind to thenon-heme iron to form the claw setting structure.

The peptide of the present invention can be obtained by, for example,enzymatical degradayion wild type NHase derived from microorganisms suchas Rhodococcus sp. N-771 (FERM P-4445). First, by treating α subunit ofwild type NHase with trypsin, the 24-residue peptide can be obtained.Then, by successively digesting this peptide with thermolysin, thenleucine aminopeptidase and carboxypeptidase M, the objective sequenceIVC₁SLC₂SC₃TAW can be obtained.

Alternatively, the peptide can be produced by a publicly known geneticengineering technique based on the desired amino acid sequence.

The present invention includes a method for imparting photoreactivity tocells by introducing the aforementioned peptide sequence of the presentinvention into at least one protein which is involved in a metabolicsystem and/or energy metabolic system of the cells. By introducing theabove peptide sequence of the present invention into a protein, aphotoreactive non-heme iron center having the claw setting, structureshown in FIG. 3 can be formed. The present invention also includes amethod for imparting photoreactivity to cells by introducing theaforementioned peptide sequence of the present invention together with anon-heme iron which is binding to the peptide into at least one proteinwhich is involved in a metabolic system and/or energy metabolic systemof the cells. The aforementioned peptide sequence of the presentinvention can be introduced into one or several kinds of proteins. Inparticular, by introducing it into several kinds of proteins, there canbe obtained advantages that metabolic rates can be controlled amongseveral pathways, and a metabolic pathway suitable for the desiredproduction can be selectively activated.

The present invention further includes a cell having an NO producingsystem and photoreactivity wherein the aforementioned peptide sequenceof the present invention is introduced into at least one protein whichis involved in a metabolic system and/or energy metabolic system of thecell. The present invention also includes a cell having an NO producingsystem and photoreactivity wherein the aforementioned peptide sequenceof the present invention is introduced together with a non-heme ironwhich is binding to the peptide (having the claw setting structure shownin FIG. 3) into at least one protein which is involved in a metabolicsystem and/or energy metabolic system of the cell. The peptide sequenceof the above the present invention can be introduced into one or severalkinds of proteins. In particular, by introducing it into several kindsof proteins, there can be obtained advantages that metabolic rates canbe controlled among several pathways, and a metabolic pathway suitablefor the desired production can be selectively activated.

As the protein to which photoreactivity is imparted, for example,enzymes having a mononuclear non-heme iron such as catechol dioxygenase,lipoxygenase and tyrosine hydroxylase can be mentioned. When theseenzymes are used, genes for them are isolated to form a recombinantexpression vector, and then a nucleotide sequence to be translated intothe peptide of the present invention is introduced into a gene regionfor the portion around the non-heme iron binding site by a recombinantDNA technique to form a expression vector for a recombinant proteinhaving a photoreactive non-heme iron center.

When it is desired to impart photoreactivity to a specific step of anenzymatic process involving several enzymes, imparting photoreactivitymay be achieved as follows. First, a recombinant gene for a proteinfunctioning in the step to which photoreactivity is desired to beimparted in which the photoreactive non-heme iron center is introducedis constructed by the method described above. Then, the recombinant geneis transferred into a cell having production process of a targetbiomolecule by using a known genetic engineering technique such ashomologous recombination to impart photoreactivity to the cell. Thus,photo-control of a production process of useful biomolecule can berealized.

A source of NO supply can be introduced by constructing an expressionvector for an NO synthase utilizing arginine as a substrate, andintroducing it into a host concurrently with the aforementioned vectorfor expressing a mutant. Because the NO synthase is activated withcalcium ion, inactivation by NO can be controlled by two kinds offactors, dark condition and calcium ion. Thus, a protein havingphotoreactivity can be prepared.

EXAMPLES

The present invention will be further explained with reference to thefollowing examples.

EXAMPLES

All manipulations were operated in the dark to avoid photodissociationof NO from the iron center. The isolation of α subunit from NHase wasperformed according to the method described in J. Biochem., 119, 407-413(1996).

The α subunit which had been isolated from inactive NHase derived fromRhodococcus sp. N-771 (FERM P-4445) was thoroughly desalted using aCentriprep-10 (Amicon) with 20 mM Tris-HCl, pH 8.0, containing 10 mMCaCl₂ and 2 mM 2-mercaptoethanol. The α subunit at a final concentrationof 2-2.5 mg/ml in 500 μl was treated with TPCK trypsin (14 μg) for 3hours at 37° C. The digests were subjected to reversed-phase HPLC with aCapcell pak C18 column (4.6×250 mm, Shiseido). The elution conditionswere as follows. Solvent A was 20 mM Tris-HCl, pH 7.5, containing 2 mM2-mereaptoethanol, and Solvent B was 20% A+80% acetonitrile. The contentof B was increased as; 0-2 min, 0%; 2-4 min, 0-20%; 4-24 min, 20-60%;and 24-26 min, 60-100%. The flow rate was 1.0 ml/min. The eluent wasmonitored by a diode array detector, HEWLETT PACKARD, HP1090 LiquidChromatography. Sequence of the product provided was determined by anamino acid sequencer. As a result, it was a single [SEQ ID NO:5]fragment composed of 24 residues of ₁₀₅Asn-₁₂₈Lys[N-V-I-V-C-S-C-T-A-W-P-I-L-G-L-P-P-T-W-Y-K]. Solvent A was changed to 5mM ammonium acetate, pH 7.5, and reversed-phase HPLC was performed underthe same condition as described above. When the amount of iron atomsbound to in the resulted peptide was determined by ICP-MS, it was foundthat one peptide bound one Fe atom.

Then, the peptide resulted above was further digested. The peptide (1.35μg) in 20 mM Tris-HCl, pH 7.5 was treated with 1 μg of thermolysin for 1hours at 37° C., and then with carboxypeptidase-Y (1 μg) and leucineaminopeptidase-M (1 μg) for 12 hours at 37° C. The digests wereseparated by reversed-phase HPLC using Capcell pak C18 column (4.6×250mm, Shiseido). The gradient condition was as follows. Solvent C was 20mM ammonium acetate, pH 7.5 and Solvent D was 20% Solvent C+80%acetonitrile. The column was equilibrated with 100% of Solvent C and alinear gradient was run from 0% of solvent D to 80% over a period of 20min at a flow rate of 0.2 ml/min. The result of the reversed-phase HPLCis shown in FIG. 1, and a UV absorption spectrum of a peak at retentiontime of 11.6 minutes in the reversed-phase HPLC is shown in FIG. 2. Inthe UV absorption spectrum of the peak at retention time of 11.6minutes, an absorption peak at 370 nm was observed, and it indicatesthat the contained fragment was associated with a nitrosylated iron. Ina photo-irradiated peptide, the aforementioned absorption peak at 370 nmcompletely disappeared.

Sequence of the resulted product was determined by an amino acidsequencer. As a result, it was a single fragment [SEQ ID NO.:6] composedof 11 residues of ₁₀₇I-V-S-L-C-S-C-T-A-₁₁₇W.

Existence of Post-translational Modification

Cysteine residues cannot be detected when sequencing was performed withan amino acid sequencer. The aforementioned 24-residue peptide of ₁₀₅Nto ₁₂₈K (₁₀₅NVIVCSLCSCTAWPILGLPPTWYK₁₂₈) contains three cysteineresidues, and the remaining 21 residues have already been identified.Therefore, also considering the result of the mass spectrometry, thepeptide fragment provided from trypsin digestion was subjected tocarboxymethylation after reduction to alkylate the cysteine residues,and then the analysis by the amino acid sequencer was attempted. As aresult, among the cysteine cluster containing three cysteines(₁₀₉C-S-L-₁₁₂CS-₁₁₄C), the 112nd cysteine still could not be detected asbefore, whereas the 109th and the 114th cysteines could be detected ascarboxymethylcysteines. At the same time, mass of this sample wasmeasured by using MALDI-TOF MS (matrix-assisted laser desorptionionization time of flight mass spectrometer) as a mass spectrometer. Asto the mass number, in addition to the molecular weight of ₁₀₅N to ₁₂₈K(2663.3), increment considered to be due to binding of twocarboxymethyls (molecular weight: 59) and further increment of 32 Dawere detected.

Identification of Cysteinesulfinic Acid

From the results of mass spectrometry and amino acid sequencing, itbecame clear that modification of 32 Da increment occurred at the 112ndcysteine. It is assumed that sulfur (32) or oxygen-molecule (16×2)corresponds to the mass number of 32. However, if it was due to sulfuratom (Cys-S₂H), it should be carboxymethylated after reduction.Therefore, the most probable modification was considered to be oxidationby two oxygen atoms. The present inventors considered that the cysteineresidue is present in the fragment as cysteinesulfinic acid (Cys-SO₂H),which corresponds to cysteine residue oxidized by two oxygen atoms, andidentified it by amino acid analysis as follows. Amino acid compositionanalysis was performed by the method established by Hayashi et al.wherein antipodes of amino acids are derivated with a fluorescentreagent, (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), and separated byreversed-phase chromatography (Hayashi et al., 1993).

In order to prevent the oxidation of cysteine residue, hydrolysis of thepeptide was performed with an enzyme (leucine aminopeptidase). However,since the 112nd, amino acid is the eighth amino acid residue from theN-terminus of the peptide, and yield will be decreased as it is moreremote from the N-terminus, the fragment was preliminarily subjected tosecondary digestion with thermolysin. The resulting digestion mixturewas purified by reversed-phase chromatography to afford severalfragments, and a fragment of the objective 8 residues of from 111stleucine to 118th proline (₁₁₁L-X-S-CM-T-A-W-₁₁₈P wherein X is a residueconsidered to be cysteinesulfinic acid, and CM is carboxymethylcysteine)was eluted at 7.8 min.

The molecular mass of the fragment obtained by the above secondarydigestion was estimated by MALDI-TOF MS, and detected as 970.7, whichwas larger than the theoretical molecular weight of 938.1 by 32 Da. Thatis, the modification of 32 Da increment still remained in this fragment.Then, it was hydrolyzed into individual amino acids with leucineaminopeptidase to perform amino acid composition analysis. As controls,D,L-cysteic acid and L-cysteinesulfinic acid were analyzed, andrespective elution time was 22.9 min (L-cysteic acid), 23.4 min(D-cysteic acid), and 26.7 min. From the area values of the peaks, itwas considered that about 39% of sulfinic acid was oxidized to cysteicacid in this measurement.

In the amino acid composition analysis of the peptide of 8 residuesobtained from the secondary digestion with thermolysin, peaks weredetected at elution times of 22.7 min and 26.2 min. From the ratio ofthe area values, it was thought that about 56% of the residue wasconverted into cysteic acid. The yield of leucine of the N-terminus ofthis fragment was 97.3 pmol, whereas the yield of cysteinesulfinic acidincluding those oxidized into cysteic acid was 99.0 pmol(cysteinesulfinic acid: 44 pmol, cysteic acid: 55 pmol).

From the results mentioned above, it has been revealed that the 112ndcysteine residue in the above peptide fragment was modified by oxidationwith two oxygen atoms into cysteinesulfinic acid (Cys-SO₂H).

Structure Determination of the Nitrosylated NHase

The crystal of the nitrosylated NHase (inactive NHase) diffracted X-rayup to high resolution, 1.7 Å resolution, and belonged to the space groupof P2₁2₁2 with cell dimensions of a=117.4 Å, b=145.6 Å and c=52.1 Å .Two αβ hetero-dimers existed in an asymmetric unit of the crystal. Thecrystal structure of the inactive NHase was determined by multipleisomorphous replacement (MIR) analysis followed by density modificationand non-crystallographic symmetry averaging. The structural model wascrystallographically refined at 1.7 Å resolution (R-factor 0.179,R-_(free)0.228). FIG. 4 shows a hetero-tetramer structure ((αβ)₂) in anasymmetric unit. The folding pattern of the inactive NHase is verysimilar to that of the photoactivated enzyme, showing that theconformation is conserved between the inactive and photoactivatedenzymes. While the inactive NHase formed a hetero-tetramer in thecrystal, sedimentation equilibrium and dynamical light scatteringmeasurements showed that the enzyme existed in a dimer-tetramerequilibrium in solution depending on its concentration. The non-hemeiron center was located at the interface between the two subunits in thehetero-dimer.

Structure of the Active Center

FIG. 5 shows the omit-annealed Fo-Fc maps of the active site region.Each residue was clearly resolved. The active site was composed of fouramino acid residues from the α subunit (αCys¹⁰⁹, αCys¹¹², αSer¹¹³,αCys¹¹⁴) and two amino acid residues from the β subunit (βArg⁵⁶,βArg¹⁴¹). The ligands to the non-heme iron atom are sulfur atoms of thethree cysteine residues (αCys¹⁰⁹, αCys¹¹², αCys¹¹⁴), main chain amidenitrogen atoms (αSer¹¹³, αCys¹¹⁴) and nitric oxide (NO). Only the siteoccupied by nitric oxide is accessible from solvent. The atomscoordinating to the iron of the inactive type were identical to those inthe photoactivated one except for the nitrogen of nitric oxide. The twosulfur atoms (S γ atoms of αCys¹¹² and αCys¹¹⁴), the two amide nitrogenatoms and the iron atom were arranged in the same plane. The length ofthe Fe—N(NO) bond in the inactive NHase was 1.65 Å, which was comparableto those in many nitrosyl iron(III) complexes. The nitric oxidecoordinated with the non-heme iron(II) in a bent configuration with aFe—N—O angle of 158.6° tilted toward the middle of αCys¹¹² and a Cys¹¹⁴.

Post-translational Modifications in the Active Center

General structure of the active center can be seen in the omit-annealedFo-Fc maps. Extra electron densities appeared around the Sγ atoms of aCys¹¹² and α Cys¹¹⁴ (FIG. 5). Since αCys¹¹² is post-translationallymodified to a cysteinesulfinic acid (Cys-SO₂H), the two additionalelectron densities around S γ of αCys¹¹² is thought to correspond to thetwo oxygen atoms of the sulfinyl group. It was assumed that αCys¹¹⁴ wasalso post-translationally modified, because there was no atom to ascribethe additional electron density close to Sγ of αCys¹¹⁴ in any knownmodels. To confirm this idea, the tryptic digests of the inactive NHasewere examined by FT-ICR MS. FIG. 7a shows the positive electrosprayionization mass spectra of a tryptic digest of inactive NHase α subunitin the neutral condition. Two signals with m/z of 1347.65 and 1383.13were assigned for doubly charged, (M+2H)²⁺, iron center peptides(αAsn¹⁰⁵-αLys¹²⁸) without and with Fe³⁺, respectively. The formercontained α Cys¹⁰⁹-S-S-αCys¹¹⁴ and αCys¹¹²-SO₂H (M_(r), =2693.31), andthe latter contained α Cys¹⁰⁹-S⁻, αCys¹¹⁴-SO⁻ and αCys¹¹²-SO²⁻(M^(r)2764.23). Assuming no post-translational modification occurred onαCys¹¹⁴, expected M_(r) of the latter (2748.24) was smaller by 16.02than the observed value. Upon addition of acetic acid, the latter signaldisappeared and the relative intensity of the former, was doubled ormore (FIG. 7b). In the acidic condition, the peptide released Fe³⁺,which was replaced by 3 protons, and a disulfide bond was formed betweenαCys-SOH¹¹⁴ and αCys-SH¹⁰⁹ as a result a water molecule is produced. Themeasured mass difference of two signals (35.48×2=70.96) was well inaccord with the calculated difference (55.93−3.03+18.01=70.92). Thus, weconcluded that αCys¹¹⁴ is modified to Cys-SOH, and the additionalelectron density corresponds to the oxygen atom of the sulfenyl group.It is considered that, since acidic condition was used for theexperiment on cysteinesulfenic acid, the presence of acid-labilecysteinesulfenic acid was overlooked.

Stabilization of Nitric Oxide by “the Claw Setting”

As shown in FIG. 6, three oxygen atoms, O δ1 of αCys-SO₂H¹¹²O δ of αCys-SOH¹¹⁴ and O γ of αSer¹¹³, were protruded from the plane containingthe iron atom by 1.5 Å like claws (of rings), and a nitric oxidemolecule was held at the center of the three “claws”. This structure isnamed as “claw setting”. These three oxygen atoms are located at adistance from the nitric oxide molecule close enough to have stronginteraction within them. The nitrosyl iron complex of the NHase, whoseiron exists in a low-spin ferric(II) state as indicated by ESR studies,is stable more than one year under aerobic condition as long as shieldedfrom light. Moreover, the nitrosylated iron center is stable even in aproteolytic fragment from αIle¹⁰⁷ to a Trp¹¹⁷, whereas the associationconstant of nitric oxide to ferric(III) irons are much smaller thanthose to ferrous(II) ones in hemeproteins, and the association isgenerally unstable in air. The extraordinary stability of the nitrosylnon-hem ferric(III) iron center in NHase having iron(III) is likely tobe due to the interaction between the nitric oxide and the oxygen atomsin the “claw setting”. On the contrary, the nitric oxide immediatelydissociates from NHase after light irradiation. Fourier transforminfrared difference spectrum showed that a local structural changeoccurs around the iron center upon light irradiation. These resultssuggest that light irradiation breaks the Fe—N(NO) bond, and thusweakens the interaction between the oxygen atoms and the nitric oxidewhich induces the structural change of the “claw setting”. Takentogether, the photoreactivity of Fe-type NHase is likely to be explainedin terms of the “claw setting”.

αCys-SO₂H¹¹² and αCys-SOH¹¹⁴ were stabilized by hydrogen bonds formedwith βArg⁵⁶ and βArg¹⁴¹ (FIG. 6). These arginine residues are conservedin all known NHases. The replacement of these residues with other aminoacids resulted in the loss of activity, and induced a significant changein the absorption spectra reflecting the electronic state of thecatalytic center (M. Tsujimura et al.). This fact suggests that the“claw setting” is also important for the enzymatic activity of NHase.

According to the present invention, there can be provided a peptide witha shorter peptide chain capable of imparting photoreactivity, which canbe easily introduced into a protein with a little risk in degradingoriginal function of the protein. It is also be possible to impartphotoreactivity to a cell by utilizing this peptide. As a result, itbecomes possible to control production of a great number of usefulbiomolecules such as amino acids, peptides, proteins, carbohydrates, andlipids by presence and absence of light irradiation.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Rhodococcus sp. N-771 <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (5)<223> OTHER INFORMATION: Amino acid at position  #5 is Xaa wherein Xaa =      an arbitrary amino acid. <400> SEQUENCE: 1Ile Val Cys Leu Xaa Ser Cys Thr Ala Trp   1               5 #                 10 <210> SEQ ID NO 2 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Rhodococcus sp. N-771 <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (3)<223> OTHER INFORMATION: Amino acid at position  #3 is Xaa wherein Xaa =      an arbitrary amino acid. <400> SEQUENCE: 2 Cys Leu Xaa Ser Cys  1               5 <210> SEQ ID NO 3 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: Rhodococcus sp. N-771 <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: Amino acids at positions #1, 2, 4, 5, 9, 10 & 11       are Xaa wherein Xaa = an ar#bitrary amino acid. <221> NAME/KEY: PEPTIDE <222> LOCATION: (6)..(8)<223> OTHER INFORMATION: Amino acid at position  #6 is Xaa wherein Xaa =      cysteinesulfinic acid. <221> NAME/KEY: PEPTIDE<222> LOCATION: (6)..(8)<223> OTHER INFORMATION: Amino acid at position  #8 is Xaa wherein Xaa =      cysteinesulfenic acid. <400> SEQUENCE: 3Xaa Xaa Cys Xaa Xaa Xaa Ser Xaa Xaa Xaa Xa #a   1               5 #                 10 <210> SEQ ID NO 4 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: Rhodococcus sp. N-771 <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: Amino acid at position  #6 is Xaa wherein Xaa =      cysteinesulfinic acid. <221> NAME/KEY: PEPTIDE<222> LOCATION: (1)..(11)<223> OTHER INFORMATION: Amino acid at position  #8 is Xaa wherein Xaa =      cysteinesulfenic acid. <400> SEQUENCE: 4Ile Val Cys Ser Leu Xaa Ser Xaa Thr Ala Tr #p   1               5 #                 10 <210> SEQ ID NO 5 <211> LENGTH: 21 <212> TYPE: PRT<213> ORGANISM: Rhodococcus sp. N-771 <400> SEQUENCE: 5Asn Val Ile Val Cys Ser Cys Thr Ala Trp Pr #o Ile Leu Gly Leu Pro  1               5  #                 10  #                 15Pro Thr Trp Tyr Lys              20 <210> SEQ ID NO 6 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Rhodococcus sp. N-771 <400> SEQUENCE: 6Ile Val Ser Leu Cys Ser Cys Thr Ala Trp   1               5 #                 10

What is claimed is:
 1. A peptide consisting of the amino acid sequenceof formula (1): X₁X₂C₁X₃X₄C₂SC₃X₅X₆X₇  (1) wherein X₁ to X₇ are anyamino acid, C₁ is cysteine, C₂ is cysteinesulfinic acid, C₃ iscysteinesulfenic acid, and S is serine.
 2. A peptide according to claim1 wherein the sequence of formula (1) is IVC₁SLC₂SC₃TAW wherein I isisoleucine, V is valine, C₁ is cysteine, C₂ is cysteinesulfenic acid, C₃is cysteiesulfenic acid, S is serine, L is leucine, T is threonin, A isalanine, and W is tryptophan.
 3. A peptide according to claim 1, whichforms a claw setting structure by binding to a non-heme iron.